Vaccine and therapeutic delivery system

ABSTRACT

The present invention relates to a new vaccine delivery system. In particular, the present invention includes compositions and methods of integrally transformed non-pathogenic, commensal bacteria that can express a nucleic acid molecule of a foreign polypeptide, wherein the nucleic acid molecule that encodes the foreign polypeptide is stably integrated into genomic DNA of the bacteria. The foreign polypeptide includes a vaccine antigen that elicits an immunogenic response, an inhibitor of a pathogen, or an immune booster or modulator.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.13/624,146, filed Sep. 21, 2012, which claims the benefit of U.S.Provisional Application No. 61/538,346, filed Sep. 23, 2011, entitled,“Mucosal Vaccination Against Infectious Diseases Delivered By AbundantCommensal Oral Bacteria” by Campos-Neto, Antonio et al.

The entire teachings of the above application are incorporated herein byreference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by a grant R01AI076425 from National Institutes of Health-National Institute ofAllergy and Infectious Disease and by a grant R01DE015931 from NationalInstitutes of Health, National Institute of Dental and CraniofacialResearch. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Most immunization strategies of current vaccines use protocols thatprime and boost the immune system using a parenteral route e.g.,intra-dermal, sub-cutaneous and intra-muscular. These means ofimmunization generate strong systemic immunity but little or no mucosalimmunity. In addition, this traditional vaccination protocol, despiteinducing both long-lived central memory T cells and short-lived effectorT cells, does not sustain a reasonable pool of the latter. These cells,which express CD4 surface antigen and recognize antigenic peptidesdisplayed on the surface of MHC II molecules are the main orchestratorsof the immune response. The short-lived T helper cells influence thefunction of important effector cells including CD8 cytotoxic T cells,antibody producing B lymphocytes and macrophages all of which areimportant in generation of a successful immune response and subsequentmemory.

The primary reason for using a mucosal route of vaccination is that mostinfections affect or start from a mucosal surface and that in theseinfections, topical application of a vaccine is often required to inducea protective immune response. Examples include gastrointestinalinfections caused by Helicobacter pylori, Vibrio cholera, andClostridium difficile; enterotoxigenic infections caused by Escherichiacoli; respiratory infections caused by Mycobacterium tuberculosis andMycoplasma pneumonia; and sexually transmitted genital infections causedby HIV, herpes simplex virus, and Chlamydia trachomatis. Therefore, aneed exists for a vaccination method in which mucosal immunity isprovided.

Yet a further need exists for using persistent and abundant commensaloral organisms to deliver antigens for mucosal immunization againstinfectious diseases. Yet a further need exists for a vaccinationprotocol that provides generation and maintenance of a significant poolof protective effector and memory T cells.

SUMMARY OF THE INVENTION

The present invention relates to integrally transformed non-pathogenic,commensal bacterium that can express one or more nucleic acid moleculesof one or more foreign polypeptides therein. The nucleic acid moleculethat encodes the foreign polypeptide is stably integrated into genomicDNA of the bacterium. The commensal bacterium used in the presentinvention includes e.g., one or more of the following: Streptococcusmitis, Streptococcus oxalis, Streptococcus sanguis, Streptococcussalivarius, Streptococcus constellatus, Lactobacillus casei,Lactobacillus fermenti, Veillonella parvula, Prevotella melaninogenica,Eikenella corrodens, Neisseria mucosa, Actinomyces odontolyticus,Fusobacterium periodonticum, Borrelia vincentii, and Actinomycesnaeslundii. The nucleic acid molecule of the foreign polypeptideincludes an antigen that elicits an immunogenic response in theindividual, a vaccine antigen, an inhibitor of a pathogen, an immunebooster, a modulator, a composition used in the treatment of a diseaseor condition, or a combination thereof. Examples of vaccine antigensinclude Mycobacterium leprae antigens, Mycobacterium tuberculosisantigens, Clostridium difficile antigens, malaria sporozoites andmerozoites, diphtheria toxoid, tetanus toxoids, Leishmania antigens,Salmonella antigens, Mycobacterium africanum antigens, Mycobacteriumintracellular antigens, Mycobacterium avium antigens, Treponemaantigens, Pertussis antigens, Herpes virus antigens, Measles virusantigens, Mumps virus antigens, Shigella antigens, Neisseria antigens,Borrelia antigens, Rabies virus antigens, polio virus antigens, humanimmunodeficiency virus antigens, snake venom antigens, insect venomantigens, hepatitis A, B, C virus, human papilloma virus antigens,Vibrio cholera, Candida albicans, Candida tropicalis, Paracoccidioidesbrasiliensis, and the like. Examples of inhibitors of a pathogen aresmall, antimicrobial peptides (e.g., defensins) that can have an effectagainst various pathogens, including M. tuberculosis, M. leprae, M.africanum, M. intracellular, M. avium; Clostridium difficile; Malaria;Diphtheria; Leishmania; Salmonella; Treponema; Pertussis; Herpes virus;Measles virus; Mumps virus; Shigella; Neisseria; Borrelia; rabies virus;polio virus; Human and human immunodeficiency virus types I and II;Hepatitis A, B, C virus, Vibrio cholera, Candida albicans, Candidatropicalis, and Paracoccidioides brasiliensis, etc. Immune boostersand/or modulators include e.g., interleukins, interferons, and cytokinessuch as IL-1, IL-2, IL-4, IL-6, IL-8, IL-12, IL-17, IL-2 IL-21 and IL-22and cytokines GMCSF, MCSF, MIP1 alpha and beta, TNF alpha and beta,IFNalpha and beta and TGFbeta. Additionally, in an embodiment of thepresent invention, the nucleic acid molecule of the foreign polypeptideis transformed into a gene that expresses a polypeptide in thecytoplasm, in the cell membrane or cell wall or exports it to the cellsurface of the bacterium. In one embodiment, the present inventionincludes utilizing S. mitis as the non-pathogenic, commensal bacterium,and the gene that expresses or secretes a polypeptide on the bacteria'scell surface is the serine-rich GspB homologue, the pullulanasepolypeptide, or both. The present invention also embodies a foreignpolypeptide that is a HIV vaccine, a tuberculosis vaccine, malariavaccine, leishmaniasis vaccine, herpes virus vaccine, hepatitis virusvaccine, or a combination thereof. Examples of the HIV antigen that canbe used with the present invention are gpl20 env, gpl40 env, gpl60 env,gag, pol, nef, vif, vpr, vpu, tat, rev, nef or HIV T20 polypeptideinhibitor. In a particular embodiment, the present invention includescommensal bacteria that have a nucleic acid sequence that encodes theHIV T20 polypeptide, the nucleic acid sequence is set forth in SEQ IDNO: 49. The present invention also includes a commensal bacterium thatencodes a foreign polypeptide that encodes the HIV T20 polypeptideaccording to SEQ ID NO: 50. The commensal bacterium, in an aspect, has anucleic acid molecule that encodes HIV T20 and is inserted into a genethat expresses or secretes a pullulanase polypeptide, to thereby obtaina gene that expresses or secretes an HIV gpl20/pullulanase fusionprotein. The nucleic acid molecule of the HIV gpl20/pullulanase fusionprotein has a sequence of SEQ ID NO: 51, and encodes a sequence of SEQID NO: 52.

In an embodiment, the integrally transformed non-pathogenic, commensalbacterium can encode a M. tuberculosis antigen having a polypeptidesequence of an amino acid sequence encoded by a nucleic acid molecule.The nucleic acid molecules can be any one of the following: SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, 43, 45, 47, or combination thereof; an amino acid sequenceencoded by a coding region of a nucleic acid molecule of SEQ ID NO: 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47, or combination thereof; an amino acid sequence encodedby a complement of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or combination thereof;an amino acid sequence encoded by a nucleic acid molecule thathybridizes to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or combination thereof underhigh stringency conditions, wherein said conditions comprise 1×SSC, 1%SDS and 0.1-2 mg/ml denatured calf thymus DNA at 65[deg.]C; and an aminoacid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 orcombination thereof.

In yet another embodiment, the integrally transformed non-pathogenic,commensal bacterium encodes a M. tuberculosis antigen. The TB antigen,for example, is encoded by one of the following nucleic acid sequences:SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 45, 47, or combination thereof; the codingregion of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or combination thereof; acomplement of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or combination thereof; asequence that hybridizes (e.g., under high stringency conditions) to SEQID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35, 37, 39, 41, 43, 45, 47, or combination thereof; and a sequence thatencodes SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or combination thereof. Thepresent invention also relates to integrally transformed bacterium forthe delivery of a foreign polypeptide to an individual with anon-pathogenic, commensal bacterium that comprises: a genomic nucleicacid molecule; a nucleic acid molecule having a nucleic acid sequencethat encodes the foreign polypeptide, wherein the nucleic acid sequenceis inserted into a gene of the genomic nucleic acid molecule whoseprotein product is expressed in the bacterium's cytoplasm or cell wallor exported to the cell surface of the bacterium; and a nucleic acidmolecule having a nucleic acid sequence for the selection of theintegrally transformed bacterium. In an aspect, the nucleic acidmolecule of the foreign polypeptide expresses an antigen that elicits animmunogenic response in the individual, a vaccine, an inhibitor of apathogen, an immune booster, a composition used in the treatment of adisease or condition, or a combination thereof. The commensal bacteriumcan be present or inoculated in an individual in an amount between about10×10<3> and about 10×10<10>. In an embodiment, the bacterium iscommensal to the oral cavity, the upper respiratory tract, or both.

The present invention also relates to systems for delivering, to anindividual, one or more foreign polypeptides that are integrallytransformed into non-pathogenic bacteria. The system includes anon-pathogenic, commensal bacterium having a genomic nucleic acidmolecule; and a nucleic acid molecule having a nucleic acid sequencethat encodes the foreign polypeptide, wherein the nucleic acid sequenceis inserted into a gene of the genomic nucleic acid molecule thatencodes a polypeptide presented on the cell wall of the bacterium. Thenucleic acid molecule of the foreign polypeptide can be an antigen thatelicits an immunogenic response in the individual, a vaccine, aninhibitor of a pathogen, an immune booster, a modulator, a compositionused in the treatment of a disease or condition, or a combinationthereof. The bacteria can include, for example, Streptococcus mitis,Streptococcus oxalis, Streptococcus sanguis, Streptococcus salivarius,Streptococcus constellatus, Lactobacillus casei, Lactobacillus fermenti,Veillonella parvula, Prevotella melaninogenica, Eikenella corrodens,Neisseria mucosa, Actinomyces odontolyticus, Fusobacteriumperiodonticum, Borrelia vincentii, and Actinomyces naeslundii. Thepolypeptide expressed by the integrally transformed non-pathogenic,commensal bacterium is described herein.

The present invention relates to methods of delivering a foreign proteinto an individual to induce mucosal immunity. The method includes thesteps of contacting the integrally transformed non-pathogenic, commensalbacterium, as described herein, with tissue of the oral cavity or upperrespiratory tract of the individual in an amount sufficient forcolonization of the bacteria. The amount of the integrally transformedbacterium that is contacted with the oral cavity or upper respiratorytract ranges, for example, between about 1×10<3> and about 1×10<10>. Themethod can further include subjecting the individual to an antibioticprior to contacting the integrally transformed non-pathogenic, commensalbacterium with the individual. The method can also include inserting anucleic acid molecule that expresses the foreign polypeptide into a geneinto the genome of the bacterium that expresses or secretes a cell-wallsurface protein. Also, the methods can further include ligating aconstruct comprising a vector having the nucleic acid molecule thatencodes the foreign polypeptide, into the gene in the genome of thebacterium that expresses or secretes a cell-wall surface protein.

The characteristics of the immune response elicited through a mucosalroute of vaccination have a number of advantages over parenteralvaccination. First, mucosal immunization results in an abundantproduction of antigen-specific IgA antibodies at the site of infection.This pathogen-specific response is important not only for the preventionof infection in vaccinated individual, but also helpful in theprevention of a healthy carrier condition and subsequent pathogentransmission to unprotected individuals. Second, in addition to IgAresponses, mucosal vaccination elicits systemic IgG responses thatcorrespond to a further defense against pathogens. Such synergisticstimulation of mucosal and systemic antibody production is important forprotection against pathogens such as HIV that can infect through bothmucosal and systemic route. Third, mucosal immunization at one siteresults in specific responses at distant sites due to mucosal lymphocytemigration and expression of homing receptors. This is important becausedefensive immunity against sexually transmitted diseases, for example,can be acquired through oral immunization. Fourth, besides serum IgG andmucosal IgA antibodies, mucosal vaccines have the ability to engage cellmediated responses including T helper and cytotoxic T cell responses,which are important for intracellular pathogen clearance. Mucosaladministration of vaccines is also associated with certain importantpractical advantages. It is non-invasive, does not require the use ofneedles, eliminates the need for expensive specialized personnel, and ischaracterized by reduced adverse effects. The development of efficaciousvaccines to serious infectious diseases such as tuberculosis and AIDShas an incalculable commercial potential. The extremely high incidence,morbidity and mortality of these diseases associated with the lack ofexisting vaccines to them, constitute by definition a solid foundationfor investment and commercialization of the final product.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a bar graph showing relative abundance of different strains ofcommensal bacteria in human oral cavity samples from 225 healthysubjects as determined by DNA-DNA hybridization assay.

FIG. 2 is a schematic illustrating the strategy for integrating genesfor HIV or M. tuberculosis antigens at the 5′ end of the S. mitispullulanase gene encoding a signal peptide that allows processing andsecretion of the M. tuberculosis and HIV antigens. The signal peptideconsists of several regions including amino terminal end region (N), ahydrophobic core (H) encoding the transmembrane domain of the peptide, asignal peptidase cleavage site (C), and an accessory sequence transportmotif (AST).

FIG. 3 is a schematic illustrating the strategy for the transformationof S. mitis. M. tuberculosis antigen 85b, HIV antigen HXBc2 orerythromycin resistance gene (ermR) were stably transformed intopulA/SMT0163 gene via homologous recombination. DNA fragments containingeither ermR alone (control), HIV/ermR, or 85b/ermR flanked by 250 bppulA 5′ and 3′ fragments were ligated into a suicide vector (pCR2.1) fortransformation and integration at the pulA locus. The table provides thetransformation efficiency of each construct by selection on erythromycincontaining plates.

FIG. 4 is a schematic illustrating the results of the genomic polymerasechain reaction (PCR) analysis demonstrating successful integration ofHIV HXBc2 gene in the S. mitis. S. mitis specific, HIV specific, andermR specific fragments were amplified using S. mitis specific primerset A/B, HIV specific primer C, and ermR-specific primer D.Electrophoresis gel image demonstrates that primers A/B generated theexpected 250 bp smt0163 PCR product, while primers C and D generated theexpected 1 kb product.

FIG. 5 is a schematic illustrating the results of the genomic PCRanalysis demonstrating successful integration of TB 85b gene in the S.mitis. Electrophoresis gel image demonstrates that pulA specific primerx and 85b specific primer y generate the expected 250 bp fragment (lanes2 and 3).

FIG. 6 contains three panels, FIG. 6(A), FIG. 6(B) and FIG. 6(C). PanelFIG. 6A is a schematic representation of HIV gpl20 gene containingC-terminal His-tag incorporated into pulA gene by homologousrecombination.

Panel FIG. 6B is a Western blot image demonstrating successfulexpression of HIV gpl20 antigen (approx. 70 kD peptide fragment)detected by anti-His-tag antibody in recombinant S. mitis cell lysate(lane 3), but not in parent S. mitis cell lysate (lane 2). His-tagged M.tuberculosis protein was used as a positive control (lanel).

Panel FIG. 6C is a Western blot image demonstrating successfulexpression of secreted HIV gpl20 antigen (approx. 70 kD peptide)detected by anti-His-tag antibody in bacterial culture supernatant (lane2 and 4), but not in the supernatant from the parent S. mitis (lane5-7). His-tagged M. tuberculosis protein was used as a positive control(lanel).

FIG. 7 contains two panels, FIG. 7(A) and FIG. 7(B). Panel FIG. 7A is atable illustrating the stability of the recombinant S. mitis vector over30 generations of replication without antibiotic. Four clonesHIVgpl20A-D were grown without antibiotic for 30 generations ofreplication. The table expresses the stability of the recombinant vectoras a number of ermR colonies over the total number of colonies streakedPanel FIG. 7B is a Western blot showing that the expression of HIV gpl20antigen remains unchanged over the 30 generations of replication.

FIG. 8 contains two panels, FIG. 8(A) and FIG. 8(B). Panels FIGS. 8 Aand B are graphical representations showing the ability of r S. mitis toelicit HIV-specific immune response in mice. Intracellular cytokinestaining in CD4+ (FIG. 8A) and CD8+ (FIG. 8B) T cells is illustrated ina series of dot plots. IFNy, TNFa, and IL-2 positive cells are detectedin HIV-1 immunized animals, but not in saline treated control animals.

FIG. 9 contains two panels, FIG. 9(A) and FIG. 9(B). Panels FIGS. 9 Aand B are graphical representations showing the ability of r S. mitis toelicit HIV-specific immune response in mice. Intracellular cytokinestaining in CD4+ (FIG. 9A) and CD8+ (FIG. 9B) T cells is illustrated ina series of dot plots. [Iota][Kappa][Nu][gamma], TNFa, and IL-2 positivecells are detected in HIV-1 immunized animals, but not in sham treatedcontrol animals.

FIG. 10 contains eight panels, FIG. 10(A), FIG. 10(B), FIG. 10(C), FIG.10(D), FIG. 10(E), FIG. 10(F), FIG. 10(G), and FIG. 10(H). Panels FIGS.10A-10G are schematics showing the nucleic acid sequences (in Bold) (SEQID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27) andcorresponding M. tuberculosis polypeptide sequences (in Bold) (SEQ IDNO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28) eluted fromMHC class 1 molecules from the macrophages of mice infected with the M.tuberculosis bacteria.

Panel FIG. 10H is a table showing tuberculosis peptide sequences, SEQ IDNos: 2, 6, 10, 14, 18, 22, and 26, eluted from MHC class 1 moleculesfrom the macrophages of mice infected with the M. tuberculosis bacteria.The figure also shows The Institute for Genomic Research (TIGR)annotation, the Swiss-Prot designation, and the protein name.

FIG. 11 is a schematic showing a peptide, SEQ ID NO: 30, in bold, foundin the urine of patients with pulmonary tuberculosis, and itscorresponding nucleic acid sequence, SEQ ID NO: 29. The figure alsoshows a DNA sequence (SEQ ID NO: 31) and its corresponding proteinsequence (SEQ ID NO: 32) which is a putative molybdopterin biosynthesisprotein that is found in M. tuberculosis and has 100% homology to SEQ IDNO: 30.

FIG. 12 is a table showing tuberculosis peptide sequences, SEQ ID Nos:34, 38, 42, and 46, found in the urine of patients with pulmonarytuberculosis and the M. tuberculosis donor protein.

FIG. 13 contains two panels, FIG. 13(A) and FIG. 13(B). Panels FIGS.13A-B are schematics showing the nucleic acid sequence (in Bold andUnderline) (SEQ ID NO: 33) and corresponding M. tuberculosis polypeptidesequence (in Bold and Underline) (SEQ ID NO: 34) found in the urine ofpatients with pulmonary tuberculosis. Also depicted are homoserine0-acetyltransferase nucleic acid and polypeptide sequences from M.tuberculosis having 100% homology to the isolated sequences (SEQ ID NOs:35 and 36, respectively), along with a table providing TIGR locus name,primary locus name, the Swiss-Prot designation, putative identification,gene symbol, TIGR cellular roles, coordinates, DNA molecule name, genelength, protein length, molecular weight, pi, percent GC, enzymeCommission #, Kingdom, and Family.

FIG. 14 contains three panels, FIG. 14(A), FIG. 14(B), and FIG. 14(C).Panels FIGS. 14A-C are schematics showing the nucleic acid sequence (inBold and Underline) (SEQ ID NO: 37) and corresponding M. tuberculosispolypeptide sequence (in Bold and Underline) (SEQ ID NO: 38) found inthe urine of patients with pulmonary tuberculosis. Also depicted areChromosome partition protein smc nucleic acid and polypeptide sequencesfrom M. tuberculosis having 100% homology to the isolated sequences (SEQID NOs: 39 and 40, respectively), along with a table providing the TIGRlocus name, primary locus name, the Swiss-Prot designation, putativeidentification, gene symbol, TIGR cellular roles, coordinates, DNAmolecule name, gene length, protein length, molecular weight, pi,percent GC, enzyme Commission #, Kingdom, and Family.

FIG. 15 contains two panels, FIG. 15(A) and FIG. 15(B). Panels FIGS. 15A-B are schematics showing the nucleic acid sequence (in Bold andUnderline) (SEQ ID NO: 41) and corresponding M. tuberculosis polypeptidesequence (in Bold and Underline) (SEQ ID NO: 42) found in the urine ofpatients with pulmonary tuberculosis. Also depicted are ornithinecarbamoyltransferase nucleic acid and polypeptide sequences from M.tuberculosis having 100% homology to the isolated sequences (SEQ ID NOs:43 and 44, respectively), along with a table providing the TIGR locusname, primary locus name, the Swiss-Prot designation, putativeidentification, gene symbol, TIGR cellular roles, coordinates, DNAmolecule name, gene length, protein length, molecular weight, pi,percent GC, enzyme Commission #, Kingdom, and Family.

FIG. 16 contains two panels, FIG. 16(A) and FIG. 16(B). Panels FIGS. 16A-B are schematics showing the nucleic acid sequence (in Bold andUnderline) (SEQ ID NO: 45) and corresponding M. tuberculosis polypeptidesequence (in Bold and Underline) (SEQ ID NO: 46) found in the urine ofpatients with pulmonary tuberculosis. Also depicted are phosphoadenosinephosphosulfate reductase nucleic acid and polypeptide sequences from M.tuberculosis having 100%) homology to the isolated sequences (SEQ IDNOs: 47 and 48, respectively), along with a table providing the TIGRlocus name, primary locus name, the Swiss-Prot designation, putativeidentification, gene symbol, TIGR cellular roles, coordinates, DNAmolecule name, gene length, protein length, molecular weight, pi,percent GC, enzyme Commission #, Kingdom, and Family.

FIG. 17 contains five panels, FIG. 17(A), FIG. 17(B), FIG. 17(C), FIG.17(D), and FIG. 17(E). Panels FIGS. 17A and B are sequence listingsshowing the nucleic acid sequence (SEQ ID NO: 53) and correspondingpolypeptide sequence (SEQ ID NO: 54) of the pula signal peptide.

Panels FIGS. 17C and D are sequence listings showing the nucleic acidsequence (SEQ ID NO: 51) and corresponding polypeptide sequence (SEQ IDNO: 52) of the pulaHIVgpl20 fusion protein.

Panel FIG. 17E is a sequence listing showing the nucleic acid sequence(SEQ ID NO: 49) and corresponding polypeptide sequence (SEQ ID NO: 50)of the HIV T20 antigen.

FIG. 18 is a Western blot showing expression of the TCdA/TCdBrecombinant protein from three different r S. mitis clones compared tothe wild type bacterium.

FIGS. 19A and B are sequence listings showing the nucleic acid sequence(SEQ ID NO: 81) and corresponding polypeptide sequence (SEQ ID NO: 82)of the TCdA/TCdB antigen.

DETAILED DESCRIPTION OF THE INVENTION

A description of preferred embodiments of the invention follows.

The present invention relates to compositions and methods for inducingmucosal immunity using an integrally transformed non-pathogeniccommensal bacterium in the oral cavity, upper respiratory tract or both.A foreign polypeptide is integrally transformed into a commensalbacterium to be used as a mucosal vaccine. Mucosal immunity is importantfor certain diseases such as tuberculosis, acquired immunodeficiencydisease syndrome (AIDS), and Clostridium difficile infections. The mainprotective mechanism of the injectable vaccines is the induction ofserum antibodies, mainly IgG, which prevent systemic spread of thepathogen and which can also, through transudation, exert a minor localprotective effect at the mucosal surfaces. Mucosal vaccination, however,induces an immune response that more closely resembles natural immunitythan the response elicited by injectable vaccine. Mucosally administeredvaccine will induce a much stronger mucosal immunity characterized bysignificantly higher local IgA antibodies and local cell mediatedimmunity than the injectable vaccine.

Prior to the present invention, formulations and vaccine deliverysystems that targeted the development of a strong mucosal immunity werenot tested. Nonetheless, the lung and other mucosal structures are animportant battleground site for the successful establishment ofinfections like tuberculosis, AIDS and other infections. Additionally,the compositions of the present invention utilize immunization protocolsthat generate and maintain a continuous supply of both effector andlong-lived memory T cells. This is an important issue because resistancemediated by effector T cells should be highly dependent on thecontinuous activation of these cells, which are capable of recognizinginvading organisms and quickly respond with production of mediators ofresistance like IFN-[gamma].

Commensal Bacteria

An important innovative aspect of the present invention is theutilization of the commensal oral organism, S. mitis, as opposed toattenuated strains of virulent bacteria (and viruses) that werepreviously tested as live antigen delivery vectors. Examples of thesesystems are Salmonella enteric serovar typhi (S. typhi) and entericserovar typhimurium (S. typhimurium), Shigella, Vibrio cholerae,Listeria monocytogenes, Mycobacterium bovis (Bacille Calmette-Guerin,BCG), and Yersinia enterocolitica. These vectors impose severalrestrictions on the downstream steps of vaccine development for humanuse including poor safety performance and poor long-term colonization.In contrast, S. mitis is an excellent commensal colonizer and abundantlypresent in the mucosal areas of the normal human oral cavity (FIG. 1)and there are no pending safety issues with the organism making it ahighly attractive for use as a delivery vector for mucosal immunization.

Commensal bacteria are those that are found in the natural flora ofliving organisms. Commensal bacteria reside mainly within mucosal sitessuch as mouth, nose, lungs, gastrointestinal and urogenital tracts.Commensal bacteria are also defined as non-pathogenic, meaning thatthese organisms do not cause disease or conditions that are harmful tothe host. Not only do they not cause harm, but also commensalpopulations are believed to keep pathogenic species in check by notallowing them to adhere to mucosal surfaces. Besides presenting physicalbarriers to invading pathogens, mucosal surfaces possess specific localimmune systems that provide an important component of protectiveimmunity. Antigen delivery directly to mucosal sites via non-pathogeniccommensal bacterial vectors provide a substantial advantage over some ofthe more conventional vaccines in that live bacteria are capable ofstimulating persistent systemic as well as local immune responses.Additionally, commensal bacteria are able to colonize the niche invadedby the pathogen and stimulate an immune response at their portal ofentry to prevent infection. Commensal bacteria are used with thecompositions and methods of the present invention as a delivery vehiclebecause they colonize well, and are easily administered, cost efficient,and well-tolerated.

S. mitis is a Gram-positive commensal bacterium (class: bacilli) thatabundantly colonizes mucosal surfaces of the oral cavity and upperrespiratory tract. S. mitis is generally not motile, does not formspores and lacks group-specific antigens. S. mitis, as well as otherstrains of commensal bacteria, possesses excellent adjuvant activity forstimulation of cell-mediated immune response, induction of long-termimmunological memory and is a very safe candidate for use in recombinantvaccines. Many strains of commensal bacteria that reside on mucosalsurfaces are excellent candidates for use as vaccine vehicles into whichgenetic material of interest can be introduced and successivelyexpressed. The present invention describes such bacterial vehicles forvaccine delivery, whereas integrally transformed non-pathogenic,commensal bacteria is used to express a peptide antigen. Examples ofsuch bacteria include Streptococcus mitis, Streptococcus oxalis,Streptococcus sanguis, Streptococcus salivarius, Streptococcusconstellatus, Lactobacillus casei, Lactobacillus fermenti, Veillonellaparvula, Prevotella melaninogenica, Eikenella corrodens, Neisseriamucosa, Actinomyces odontolyticus, Fusobacterium periodonticum, Borreliavincentii, and Actinomyces naeslundii.

Foreign Polypeptides

Recombinant commensal bacteria of the present invention are engineeredto express foreign polypeptides either on their cell-wall surface or inthe secreted form. As used herein, the term “foreign polypeptide”encompasses amino acid chain of any length, including full lengthproteins (i.e., antigens), wherein their sequence is not encoded by theendogenous bacterial DNA. Foreign polypeptide, in this case, can be anyforeign antigen that elicits an immunogenic response including but notlimited to a vaccine antigen, an immune booster or modulator of theimmune response, pathogen inhibitor, or any combination of the above.Vaccine antigen used with the compositions of present invention refersto any foreign polypeptide that elicits a protective cell mediatedand/or humoral immune response and results in a long-term immunologicalmemory. There are several types of antigen-based vaccines includingpurified and recombinant vaccines. Purified antigen vaccines, alsoreferred to as subunit vaccines, are composed of molecules purifieddirectly from the pathogenic organisms. Purified antigen vaccinesidentify molecules that generate a protective immune response includingstructural proteins, polysaccharides, and chemically inactivated orattenuated bacterial toxins (exotoxins). Examples of purified antigenvaccines include Streptococcus pneumoniae and Neisseria meningitidispolysaccharides, and Clostridium difficile and Clostridium tetanitoxins. Most purified antigen vaccines require the use of adjuvant toelicit a strong immune response and multiple immunizations are oftenrequired.

Recombinant antigen vaccines are composed of immunogenic proteinsproduced by genetic engineering. DNA encoding for an immunogenic proteinof a pathogen can be inserted into either bacteria, yeast, viruses whichinfect mammalian cells, or by transfection of mammalian cells. The cellswill then produce the protein endogenously and the protein can beharvested.

Recombinant vector vaccines utilize the attenuated versions of certainmicrobes as recombinant vectors to express target antigens from otherpathogens. The major advantage of recombinant vector vaccines is theability to generate both humoral and cell-mediated immune responsesresulting in stronger, longer-lasting protection. The present inventionencompasses methods for the development of recombinant vector vaccinesbased on commensal bacteria stably transformed with constructs encodingthe following: M. leprae antigens, M. tuberculosis antigens, M.africanum antigens, M. tuberculosis intracellular antigens, M. aviumantigens, C. difficile antigens, Treponema antigens, Pertussis antigens,Herpes virus antigens, Measles virus antigens, Mumps virus antigens,Shigella antigens, Neisseria antigens, Borrelia antigens, Rabies virusantigens, polio virus antigens, human immunodeficiency virus antigens,snake venom antigens, insect venom antigens, hepatitis A, B, C virus,human papilloma virus antigens, Vibrio cholera, and fungus organismssuch as Candida albicans, Candida tropicalis, Paracoccidioidesbrasiliensis etc. Specific examples include TB and HIV antigenic peptidesequences listed below (SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or combinationsthereof, shown in FIGS. 10-16) and HIV sequences (SEQ ID NO:50, 52 shownin FIG. 17) and C. difficile sequences (SEQ ID NO: 80 shown in FIG. 19).

An Inhibitor of a Pathogen

The present invention also describes the development of therapeuticvaccines based on recombinant commensal bacteria expressing a pathogeninhibiting peptide. An inhibitor of pathogen can include any peptidethat prevents or reduces the host infection by pathogen by either of thefollowing means inhibition of pathogen interaction with host receptornecessary for infection, inhibition of pathogen replication at the siteof entry into the host, inhibition of the assembly of newly formedpathogenic entities (relevant for viral pathogens), and neutralizationof toxins released by pathogenic organisms (relevant for bacterialpathogens). One example described in the present invention includes apotent HIV inhibitor peptide, T20, which is a 36 amino acid peptide,expressed by a recombinant S. mitis, that acts as a decoy a-helix andprevents HIV-1 infection by disrupting the assembly of the six-helixbundle viral fusion apparatus by mimicking the heptad repeat 2 (HR2)oligomerization domain of the gp41 envelope glycoprotein. Additionally,present invention includes a foreign polypeptide that includes HIVantigen as follows: gpl20 env, gpl40 env, gpl60 env, gag, pol, nef, vif,vpr, vpu, tat, rev, nef or HIV T20. Additional sequences can be designedto express inhibitors of the following pathogens: Mycobacteriumtuberculosis; Mycobacterium leprae; Mycobacterium africanum;Mycobacterium intracellulare; Mycobacterium avium; Clostridiumdifficile; Malaria; Diphtheria; Leishmania; Salmonella; Treponema;Pertussis; Herpes virus; Measles virus; Mumps virus; Shigella;Neisseria; Borrelia; rabies virus; polio virus; Human immunodeficiencyvirus type I and II; Hepatitis A, B, C virus, Vibrio cholera; and fungusorganisms such as Candida albicans, Candida tropicalis, Paracoccidioidesbrasiliensis etc. TB vaccines and Mycobacterium vaccine antigens aredescribed in U.S. Pat. No. 7,968,694.

An Immune Booster and a Modulator

The present invention describes the use of additional sequences encodingimmune booster and modulator for generation of recombinant commensalbacteria based vaccine. Several classes of immune boosters andmodulators can be defined, including: interferons, interleukins, andother cytokines Certain mucosal vaccine antigens do not have the abilityto induce a potent adaptive immune response and significantimmunoglobulin production and therefore an appropriate adjuvant may berequired. Cytokines are powerful modulators of the immune response thathave the ability to trigger both adaptive and innate immune response bystimulating Th1 and Th2 responses, maturation of antigen presentingcells, and induction of NK cells and CTLs. Therefore, cytokines can beused as potent mucosal vaccine adjuvants for enhancing the immuneresponse against infectious pathogens. Interferons play an importantrole in the defense against viral pathogens.

They are part of innate immune response and often induced at an earlystage during viral infection. Interferons typically stimulate the cellsof the innate immune system to increase the expression of MHC moleculesand promote more efficient antigen presentation to helper T cells.Therefore, interferons can also be useful adjuvants in the developmentof recombinant mucosal vaccines. Examples of immune boosters andmodulators include: Interleukins, interferons, and cytokines such asIL-1, IL-2, IL-4, IL-6, IL-8, IL-12, IL-17, IL-2 IL-21 and IL-22 andcytokines GMCSF, MCSF, MIP1 alpha and beta, TNF alpha and beta, IFNalphaand beta and TGFbeta.

Integrative Transformation

Bacterial transformation is defined as introduction of foreign geneticmaterial in the form of a DNA plasmid or fragment into bacterial cellsresulting in incorporation and expression of exogenous genes by thetransformed bacteria. Bacteria that can be transformed are referred toas “competent.” Different methods exist for the induction of bacterialcompetence including calcium chloride based transformation andelectroporation. The mechanism of competence induction is based on theability to create pores in the cell membrane of the bacteria, whichallows passive uptake of plasmid DNA into bacterial cells. Plasmids aresmall circular pieces of DNA of about 2,000 to 10,000 base pairs thatmay or may not contain important genetic information for the growth ofbacteria. The main components of a plasmid include the origin ofreplication, multiple cloning site, and antibiotic resistance gene.Recombinant plasmid vectors can be created by introducing a gene ofinterest into a multiple cloning site followed by selection onantibiotic containing growth media. Transformed bacterial cellscontaining newly introduced plasmid become antibiotic resistant andsuccessfully survive on antibiotic-rich media. A self-replicatingrecombinant vector, in this case, is propagated in the bacteriaindependently of the bacterial genome.

In contrast, integrative transformation describes the stableintroduction of the whole or part of a recombinant vector into thebacterial genome itself. A construct of interest can be engineered toreplace one allele in the genome without affecting any other locus ofthe bacterial chromosome. The method of choice for a stable introductionof a construct into the bacterial genome is known as “homologousrecombination.” DNA sequence of the gene to be replaced is known inorder to engineer a construct for homologous recombination. The sequenceof an engineered construct includes some flanking DNA on both sides thatis identical in sequence to the targeted locus.

The present invention encompasses the construction of a vector forstable integration of the genes coding, for example for M. tuberculosisAg85b, HIV1 HXBc2 env-His-tag antigens, and C. difficile TcdA and TcdBantigens into the genome of S. mitis. These genes were integrated intothe signal sequence of pulA gene, coding for the pullulanase enzyme.Pullulanase is an amylo lytic exoenzyme that is produced as acell-surface anchored lipoprotein by Gram-positive bacteria. PulA wasselected as an integration site because the gene has a strong promoterand encodes localization motifs for the gene product;

furthermore, pulA is not essential for bacterial growth. The recombinantvector was generated by incorporating sequences encoding M. tuberculosisand HIV antigens preceded by an accessory sequence transport motif (AST)next to the peptidase cleavage site (C) (FIG. 2). For stable expression,the M. tuberculosis, HIV antigen, and C. difficile TcdA and TcdB antigenencoding genes along with ermR cassette, flanked on both sides by 250 bppulA 5′ and 3′ fragments, were integrated via homologous recombinationinto the pulA gene of S. mitis (FIGS. 3-5). Another vector wasconstructed in the same manner to incorporate HIV Env gene into S. mitisby homologous recombination (FIG. 6).Methods for Immunizing

Once a foreign gene has been integrated into the genome of the commensalbacterium, the integrant bacteria, such as those of the presentinvention, can be introduced to an individual in need thereof. Themethods of the present invention include methods for vaccinating anindividual by inoculating the individual with the integrant commensalbacteria having the foreign polypeptide. Inoculating procedures caninclude swabbing, spraying, inhaling or otherwise introducing theintegrated commensal bacteria to the oral cavity or upper respiratorysystem. The integrated commensal bacteria used in the invention can beadministered nasally, topically, or by inhalation.

The commensal bacteria should be administered/inoculated in amountssufficient to result in colonization of the bacteria in the oral cavity,upper respiratory tract, or both. The composition can be administered ina single dose or in more than one dose over a period of time so that thebacteria colonize and the foreign polypeptide confers the desired effect(e.g., is administered to the individual and the individual obtainsimmunity).

The actual effective amounts of integrant commensal bacteria of thepresent invention, delivered with or without a carrier, can varyaccording to the specific composition being utilized, the mode ofadministration and the age, weight and condition of the patient. Forexample, as used herein, an effective amount of bacteria is an amountwhich allows the bacteria to colonize in the oral cavity and/or upperrespiratory system. Dosages for a particular individual patient can bedetermined by one of ordinary skill in the art using conventionalconsiderations (e.g. by means of an appropriate, conventionalpharmacological protocol). In an embodiment, the amount of the commensalbacteria of the present invention used to inoculate an individual rangesin an amount between about 10×10<3> and about 10×10<10>.

For enteral or mucosal application (including via oral and nasalmucosa), particularly suitable are liquids, drops, or suppositories. Asyrup, elixir or the like can be used wherein a sweetened vehicle isemployed. Liposomes, microspheres, and microcapsules are available andcan be used.

Pulmonary administration can be accomplished, for example, using any ofvarious delivery devices known in the art such as an inhaler. See. e.g.,S. P. Newman (1984) in Aerosols and the Lung, Clarke and Davis (eds.),Butterworths, London, England, pp. 197-224; PCT Publication No. WO92/16192; PCT Publication No. WO 91/08760.

The administration of the commensal bacteria (integrants) of the presentinvention can occur simultaneously or sequentially in time inconjunction with other conventional vaccination systems and protocols orin isolation. A commensal bacterium and/or pharmaceutical composition asdescribed above can be administered simultaneously with or sequentiallywith an immune enhancer, or other compound known in the art that wouldbe administered with such a vaccine. The compound can be administeredbefore, after or at the same time as the integrated commensal bacteriaof the present invention. Thus, the term “co-administration” is usedherein to mean that the integrated commensal bacteria and the additionalcompound (e.g., immune stimulating compound) will be administered attimes to achieve an immune response, as described herein. The methods ofthe present invention are not limited to the sequence in which thecompounds are administered, so long as the compound is administeredclose enough in time to produce the desired effect.

Routes and frequency of administration of the inventive pharmaceuticalcompositions and vaccines, as well as dosage, will vary from individualto individual or from system to system. In general, the pharmaceuticalcompositions and vaccines can be administered intranasally or orally. Anexample of the protocol for inoculating an individual with integratedcommensal bacteria is as follows: inoculate three times, one month apartbetween inoculations.

The compositions of the present invention are preferably formulated aseither pharmaceutical compositions or as vaccines for in the inductionof protective immunity against the foreign polypeptide in a patient. Apatient can be afflicted with a disease, or can be free of detectabledisease and/or infection. In other words, protective immunity can beinduced to prevent, reduce the severity of, or treat the diseaseassociated with the foreign bacteria.

In one embodiment, pharmaceutical compositions of the present inventioncomprise one or more of the integrated commensal bacteria, and aphysiologically acceptable carrier. Similarly, integrated commensalbacteria comprise one or more the above polypeptides and a non-specificimmune response enhancer, such as an adjuvant or a liposome (into whichthe polypeptide is incorporated).

The integrant commensal bacteria of the present invention can beadministered with or without a carrier. The terms “pharmaceuticallyacceptable carrier” or a “carrier” refer to any generally acceptableexcipient or drug delivery composition that is relatively inert andnon-toxic. Exemplary carriers include sterile water, salt solutions(such as Ringer's solution), alcohols, gelatin, talc, viscous paraffin,fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, calciumcarbonate, carbohydrates (such as lactose, sucrose, dextrose, mannose,albumin, starch, cellulose, silica gel, polyethylene glycol (PEG), driedskim milk, rice flour, magnesium stearate, and the like. Suitableformulations and additional carriers are described in Remington'sPharmaceutical Sciences, (17th Ed., Mack Pub. Co., Easton, Pa.). Suchpreparations can be sterilized and, if desired, mixed with auxiliaryagents, e.g., lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, coloring,preservatives and/or aromatic substances and the like which do notdeleteriously react with the active compounds. Typical preservatives caninclude, potassium sorbate, sodium metabisulfite, methyl paraben, propylparaben, thimerosal, etc. The compositions can also be combined wheredesired with other active substances, e.g., enzyme inhibitors, to reducemetabolic degradation. A carrier (e.g., a pharmaceutically acceptablecarrier) is preferred, but not necessary to administer the compound.

The composition can be a liquid solution, suspension, emulsion,sustained release formulation, gel, mist, or spray. The method ofadministration can dictate how the composition will be formulated. Forexample, the composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides.

Measuring the Immunogenicity of the Vaccine

Once the commensal bacteria are administered to the individual, anindividual can be assessed to determine if the vaccine provided animmunogenic response to the foreign polypeptide. The efficacy of amucosal immunization can be measured by determining the immunogenicresponse of the person who received the vaccine. The foreign peptidesdescribed herein expressed on the surface of recombinant S. mitis (r S.mitis) have the ability to elicit an immune response. More precisely,administration of r S. mitis results in activation of the components ofcell mediated immunity, specifically CD4+ and CD8+ T cells, NK cells, Bcells and/or macrophages, leading to proliferation and cytokineproduction including but not limited to IFNy, TNFa and IL-2 in animmunized individual. Immunological evaluation methods for cell mediatedimmune response include antigen specific stimulation of cells followedby measurements of secreted cytokines by enzyme-linked immunosorbentassay (ELISA) or multiplex bead assay, measurement of intracellularcytokine levels by flow cytometry, as well as measurements of cellproliferation.

The ability of a polypeptide or bacterial lysate to stimulate thesecretion of cytokines can be evaluated by contacting the cells with thepolypeptide and measuring cytokine levels in the supernatant. Ingeneral, the amount of polypeptide that is sufficient for the evaluationof about 10<5> cells ranges from about 10 ng/mL to about 100 [mu]g/mLand preferably is about 10 [mu]g/mL. The polypeptide can, but need not,be immobilized on a solid support, such as a bead or a biodegradablemicrosphere, such as those described in U.S. Pat. Nos. 4,897,268 and5,075,109. The incubation of polypeptide with the cells is typicallyperformed at 37[deg.] C. for about six days. Following incubation withpolypeptide, the culture supematants are assayed for IFN-[gamma] (and/orTNFa and IL-2), which can be evaluated by methods known to those ofordinary skill in the art, such as an enzyme-linked immunosorbent assay(ELISA). In general, a polypeptide that results in the production of atleast 50 pg of interferon [gamma] per mL of cultured supernatant(containing 10<4>-10<5> T cells per mL) is considered able to stimulatethe production of IFN-[gamma]. A polypeptide that stimulates theproduction of at least 100 pg/mL of TNF [alpha], and/or at least 10 U/mLof IL-2, per 10<5> T cells (or per 3×10<5> PBMC) is considered able tostimulate the production of TNF a and/or IL-2.

Intracellular cytokine staining is a flow cytometric technique that candetect single cell expression of cytokines and allows simultaneousdetection, quantitation, and phenotypic characterization ofantigen-specific T cells in PBMC. In this multiparametric flow cytometrybased method, antigen specific T cells are identified based on theirintracellular accumulation of a cytokine in conjunction with theircharacteristic CD4+ and CD8+ T cells, activation, effector, memory andmucosal homing surface markers following antigenic stimulation of PBMC.Intracellular cytokine staining assay is performed on PBMC isolated fromwhole blood of immunized individual by Ficoll gradient centrifugation.Cells are then restimulated by contact with polypeptide (e.g., animmunogenic antigen, or a portion or other variant thereof) orrecombinant bacterial lysate for 6-10 hours at 37 C. Restimulated cellsare then permeabilized and contacted with fluorescently labeledmonoclonal antibodies against interferon [gamma], TNFa, IL-2, andgranzyme) as well as antibodies against surface markers characteristicfor particular cell type. Percent of IFN-[gamma], TNFa, and IL-2positive cells is then determined using appropriate software (FIGS. 8,9). A polypeptide that stimulates statistically significant increase inthe number of IFN-[gamma], TNFa, and IL-2 positive cells when comparedto control is considered able to stimulate cytokine production.

Polypeptides and their Function

The present invention relates to polypeptide molecules encoded by therecombinant commensal bacteria including antigenic portions of TB, HIV,Plasmodium, Leishmania, herpes virus, or hepatitis virus. The presentinvention includes polypeptide molecules that are encoded by the nucleicacid of the recombinant commensal bacteria and which contain thesequence of any one of the antigenic TB amino acid sequences (e.g., SEQID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 48, or combinations thereof, shown FIGS. 10-16)or HIV sequences (e.g., SEQ ID NO: 50, 52 shown in FIG. 17), or any ofthe above fused to the pulA signal peptide. The present invention alsopertains to polypeptide molecules that are encoded by nucleic acidsequence of the transformed commensal bacteria and that include thenucleic acid sequences of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or combinationsthereof.

As used herein, the term “polypeptide” encompasses amino acid chains ofany length, including full length proteins (i.e., antigens), wherein theamino acid residues are linked by covalent peptide bonds. Thus, apolypeptide comprising an immunogenic portion of any of the above namedantigens can consist entirely of the immunogenic portion, or can containadditional sequences. The additional sequences can be derived from thenative antigens or can be heterologous, and such sequences can (but neednot) be immunogenic.

The compositions and methods of the present invention also encompassvariants of the above polypeptides and DNA molecules. A polypeptide“variant,” as used herein, is a polypeptide that differs from therecited polypeptide only in conservative substitutions and/ormodifications, such that the therapeutic, antigenic and/or immunogenicproperties of the polypeptide are retained. A variant of a specificantigen will therefore stimulate cell proliferation and/or interferonproduction in Th1 cells raised against that specific antigen.Polypeptide variants preferably exhibit at least about 70%, morepreferably at least about 90% and most preferably at least about 95%homology to the identified polypeptides. For polypeptides withimmunoreactive properties, variants can, alternatively, be identified bymodifying the amino acid sequence of one of the above polypeptides, andevaluating the immunoreactivity of the modified polypeptide. Suchmodified sequences can be prepared and tested using, for example, therepresentative procedures described herein.

As used herein, a “conservative substitution” is one in which an aminoacid is substituted for another amino acid that has similar properties,such that one skilled in the art of peptide chemistry would expect thesecondary structure and hydropathic nature of the polypeptide to besubstantially unchanged.

Variants can also, or alternatively, contain other modifications,including the deletion or addition of amino acids that have minimalinfluence on the antigenic properties, secondary structure andhydropathic nature of the polypeptide. For example, a polypeptide can beconjugated to a signal (or leader) sequence at the N-terminal end of theprotein which co-translationally or post-translationally directstransfer of the protein. The polypeptide can also be conjugated to alinker or other sequence for ease of synthesis, purification oridentification of the polypeptide (e.g., poly-His), or to enhancebinding of the polypeptide to a solid support. For example, apolypeptide can be conjugated to an immunoglobulin Fc region.

The present invention also encompasses proteins and polypeptides,variants thereof, or those having amino acid sequences analogous to theamino acid sequences of antigenic polypeptides described herein. Suchpolypeptides are defined herein as antigenic analogs (e.g., homologues),or mutants or derivatives. “Analogous” or “homologous” amino acidsequences refer to amino acid sequences with sufficient identity of anyone of the amino acid sequences so as to possess the biological activity(e.g., the ability to elicit a protective immune response to antigenexpressed by commensal bacteria) of any one of the native polypeptides.For example, an analog polypeptide can be produced with “silent” changesin the amino acid sequence wherein one, or more, amino acid residuesdiffer from the amino acid residues of any one of the protein, yet stillpossesses the function or biological activity of the antigen peptide.Examples of such differences include additions, deletions orsubstitutions of residues of the amino acid sequence of antigenpeptides. Also encompassed by the present invention are analogouspolypeptides that exhibit greater, or lesser, biological activity of anyone of the proteins of the present invention. Such polypeptides can beexpressed by mutating (e.g., substituting, deleting or adding) nucleicacid residues of any of the sequences described herein. Such mutationscan be performed using methods described herein and those known in theart. In particular, the present invention relates to homologouspolypeptide molecules having at least about 70% (e.g., 75%, 80%, 85%,90% or 95%) identity or similarity with SEQ ID NO: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,or combination thereof. Percent “identity” refers to the amount ofidentical nucleotides or amino acids between two nucleotides or aminoacid sequences, respectfully. As used herein, “percent similarity”refers to the amount of similar or conservative amino acids between twoamino acid sequences.

Homologous polypeptides can be determined using methods known to thoseof skill in the art. Initial homology searches can be performed at NCBIagainst the GenBank, EMBL and SwissProt databases using, for example,the BLAST network service. Altschuler, S. F., et al, J. Mol. Biol,215:403 (1990), Altschuler, S. F., Nucleic Acids Res., 25:3389-3402(1998). Computer analysis of nucleotide sequences can be performed usingthe MOTIFS and the FindPatterns subroutines of the Genetics ComputingGroup (GCG, version 8.0) software. Protein and/or nucleotide comparisonswere performed according to Higgins and Sharp (Higgins, D. G. and Sharp,P. M., Gene, 73:237-244 (1988) e.g., using default parameters).

Additionally, the individual isolated polypeptides of the presentinvention are biologically active or functional and play various rolesin bacteria as well. For example, an isolated polypeptide, such as SEQID NO: 6 is a glutamine-transport transmembrane protein ABC transporter.Likewise, SEQ ID NO: 22 is a cationic amino acid transport integralmember protein, and SEQ ID NO: 26 is a cationic transporting P-typeATPase. SEQ ID NO: 32, is a molybdopterin biosynthesis protein. Thepresent invention includes fragments of these isolated amino acidsequences that still possess the function or biological activity of thesequence. For example, polypeptide fragments comprising deletion mutantsof the antigenic TB proteins can be designed and expressed by well-knownlaboratory methods. Fragments, homologues, or analogous polypeptides canbe evaluated for biological activity, as described herein.

The present invention also encompasses biologically active derivativesor analogs of the above described antigenic polypeptides, referred toherein as peptide mimetics. Mimetics can be designed and produced bytechniques known to those of skill in the art. (see e.g., U.S. Pat. Nos.4,612,132; 5,643,873 and 5,654,276). These mimetics can be based, forexample, on a specific amino acid sequence and maintain the relativeposition in space of the corresponding amino acid sequence. Thesepeptide mimetics possess biological activity similar to the biologicalactivity of the corresponding peptide compound, but possess a“biological advantage” over the corresponding antigenic TB amino acidsequence with respect to one, or more, of the following properties:solubility, stability and susceptibility to hydrolysis and proteolysis.

Methods for preparing peptide mimetics include modifying the N-terminalamino group, the C-terminal carboxyl group, and/or changing one or moreof the amino linkages in the peptide to a non-amino linkage. Two or moresuch modifications can be coupled in one peptide mimetic molecule.Modifications of peptides to produce peptide mimetics are described inU.S. Pat. Nos. 5,643,873 and 5,654,276. Other forms of the antigenicpolypeptides, encompassed by the present invention, include those whichare “functionally equivalent.” This term, as used herein, refers to anynucleic acid sequence and its encoded amino acid, which mimics thebiological activity of the polypeptides and/or functional domainsthereof.

Nucleic Acid Sequences, Plasmids, Vectors and Host Cells

The present invention, in one embodiment, includes an isolated nucleicacid molecule integrated into the commensal bacteria, having a sequenceof SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 45, 47, or combinations thereof. See FIGS.10-17. The present invention includes sequences as recited in FIGS.10-17, as well as the coding regions thereof.

As used herein, the terms “DNA molecule” or “nucleic acid molecule”include both sense and anti-sense strands, cDNA, genomic DNA,recombinant DNA, RNA, and wholly or partially synthesized nucleic acidmolecules. A nucleotide “variant” is a sequence that differs from therecited nucleotide sequence in having one or more nucleotide deletions,substitutions or additions. Such modifications can be readily introducedusing standard mutagenesis techniques Nucleotide variants can benaturally occurring allelic variants, or non-naturally occurringvariants. Variant nucleotide sequences preferably exhibit at least about70%, more preferably at least about 80%> and most preferably at leastabout 90%> homology to the recited sequence. Such variant nucleotidesequences will generally hybridize to the recited nucleotide sequenceunder stringent conditions. In one embodiment, “stringent conditions”refers to prewashing in a solution of 6×SSC, 0.2%> SDS; hybridizing at65<0> Celsius, 6×SSC, 0.2%> SDS overnight; followed by two washes of 30minutes each in 1×SSC, 0.1% SDS at 65<0> C. and two washes of 30 minuteseach in 0.2×SSC, 0.1% SDS at 65 [deg.] C.

The present invention also encompasses genomic commensal DNA havingisolated nucleic acid sequences that encode TB and HIV polypeptides, andin particular, those which encode a polypeptide molecule having an aminoacid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or combinations thereof.These nucleic acid sequences encode polypeptides that stimulate aprotective immunogenic response and/or are involved the functionsfurther described herein. As used herein, an “isolated” gene ornucleotide sequence which is not flanked by nucleotide sequences whichnormally (e.g., in nature) flank the gene or nucleotide sequence (e.g.,as in genomic sequences) and/or has been completely or partiallypurified from other transcribed sequences (e.g., as in a cDNA or R Alibrary). Thus, an isolated gene or nucleotide sequence can include agene or nucleotide sequence which is synthesized chemically or byrecombinant means. Nucleic acid constructs contained in a vector areincluded in the definition of “isolated” as used herein. Also, isolatednucleotide sequences include recombinant nucleic acid molecules andheterologous host cells, as well as partially or substantially orpurified nucleic acid molecules in solution. In vivo and in vitro RNAtranscripts of the present invention are also encompassed by “isolated”nucleotide sequences. Such isolated nucleotide sequences are useful forthe manufacture of the encoded antigenic TB polypeptide, for detectingthe presence (e.g., by PCR amplification and DNA sequencing) orexpression (e.g., by reverse transcription (RT)-PCR and DNA sequencing)of related genes in cells or tissue, and for gene mapping (e.g., by insitu hybridization).

The antigenic nucleic acid sequences of the present invention includehomologous nucleic acid sequences. “Analogous” or “homologous” nucleicacid sequences refer to nucleic acid sequences with sufficient identityof any one of the TB nucleic acid sequences, such that once encoded intopolypeptides, they possess the biological activity of any one of theantigenic polypeptides described herein. For example, an analogousnucleic acid molecule can be produced with “silent” changes in thesequence wherein one, or more, nucleotides differs from the nucleotidesof any one of the polypeptides described herein, yet, once encoded intoa polypeptide, still possesses its function or biological activity.Examples of such differences include additions, deletions orsubstitutions. Also encompassed by the present invention are nucleicacid sequences that encode analogous polypeptides that exhibit greater,or lesser, biological activity of the TB proteins of the presentinvention. In particular, the present invention is directed to nucleicacid molecules having at least about 70% (e.g., 75%, 80%, 85%, 90% or95%) identity with SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or combinations thereof.

The nucleic acid molecules of the present invention, including the fulllength sequences, the partial sequences, functional fragments andhomologues, once encoded into polypeptides, elicit a specificimmunogenic TB response, or has the function of the polypeptide, asfurther described herein. The homologous nucleic acid sequences can bedetermined using methods known to those of skill in the art, and bymethods described herein including those described for determininghomologous polypeptide sequences.

Also encompassed by the present invention are nucleic acid sequences,DNA or R A, which are substantially complementary to the DNA sequencesencoding the antigenic polypeptides of the present invention. Suchsequences can be used to design PCR primers to amplify and isolatehomologous DNA coding sequences from related Mycobacteria spp. Thepresent invention includes sequences those that specifically hybridizeunder conditions of stringency known to those of skill in the art. Asdefined herein, substantially complementary means that the nucleic acidneed not reflect the exact sequence of the TB sequences, but must besufficiently similar in sequence to permit hybridization with TB nucleicacid sequence under high stringency conditions. For example,non-complementary bases can be interspersed in a nucleotide sequence, orthe sequences can be longer or shorter than the TB nucleic acidsequence, provided that the sequence has a sufficient number of basescomplementary to the TB sequence to allow hybridization therewith.Conditions for stringency are described in e.g., Ausubel, F. M., et al,Current Protocols in Molecular Biology, (Current Protocol, 1994), andBrown, et al, Nature, 366:575 (1993); and further defined in conjunctionwith certain assays.

In another embodiment, the present invention includes genomic DNA thathas nucleic acid molecules (e.g., probes or primers) that hybridize tothe antigenic sequences, SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or combinationsthereof under high or moderate stringency conditions. In one aspect, thepresent invention includes molecules that hybridize or contain at leastabout 20 contiguous nucleotides or longer in length (e.g., 50, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500,1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700,2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900,or 4000). Such molecules hybridize to one of the TB nucleic acidsequences under high stringency conditions. The present inventionincludes such molecules and those that encode a polypeptide that has thefunctions or biological activity described herein.

Typically the nucleic acid probe comprises a nucleic acid sequence (e.g.SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 45, 47, or combinations thereof) and is ofsufficient length and complementarity to specifically hybridize to anucleic acid sequence that encodes an antigenic polypeptide. Forexample, a nucleic acid probe can be at least about 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80% or 90% the length of the TB nucleic acidsequence. The requirements of sufficient length and complementarity canbe easily determined by one of skill in the art. Suitable hybridizationconditions (e.g., high stringency conditions) are also described herein.Additionally, the present invention encompasses fragments of thepolypeptides of the present invention or nucleic acid sequences thatencodes a polypeptide wherein the polypeptide has the biologicallyactivity of the polypeptides recited herein.

Such fragments are useful as probes for assays described herein, and asexperimental tools, or in the case of nucleic acid fragments, asprimers. A preferred embodiment includes primers and probes whichselectively hybridize to the nucleic acid constructs encoding any one ofthe recited polypeptides. For example, nucleic acid fragments whichencode any one of the domains described herein are also implicated bythe present invention.

Stringency conditions for hybridization refers to conditions oftemperature and buffer composition which permit hybridization of a firstnucleic acid sequence to a second nucleic acid sequence, wherein theconditions determine the degree of identity between those sequenceswhich hybridize to each other. Therefore, “high stringency conditions”are those conditions wherein only nucleic acid sequences which are verysimilar to each other will hybridize. The sequences can be less similarto each other if they hybridize under moderate stringency conditions.Still less similarity is needed for two sequences to hybridize under lowstringency conditions. By varying the hybridization conditions from astringency level at which no hybridization occurs, to a level at whichhybridization is first observed, conditions can be determined at which agiven sequence will hybridize to those sequences that are most similarto it. The precise conditions determining the stringency of a particularhybridization include not only the ionic strength, temperature, and theconcentration of destabilizing agents such as formamide, but alsofactors such as the length of the nucleic acid sequences, their basecomposition, the percent of mismatched base pairs between the twosequences, and the frequency of occurrence of subsets of the sequences(e.g., small stretches of repeats) within other non-identical sequences.Washing is the step in which conditions are set so as to determine aminimum level of similarity between the sequences hybridizing with eachother. Generally, from the lowest temperature at which only homologoushybridization occurs, a 1% mismatch between two sequences results in a1[deg.] C. decrease in the melting temperature (Tm) for any chosen SSCconcentration. Generally, a doubling of the concentration of SSC resultsin an increase in the Tm of about 17[deg.] C. Using these guidelines,the washing temperature can be determined empirically, depending on thelevel of mismatch sought. Hybridization and wash conditions areexplained in Current Protocols in Molecular Biology (Ausubel, F. M. etal, eds., John Wiley & Sons, Inc., 1995, with supplemental updates) onpages 2.10.1 to 2.10.16, and 6.3.1 to 6.3.6.

High stringency conditions can employ hybridization at either (1) 1×SSC(10×SSC=3M NaCl, 0.3M Na3-citrate . . . 2H20 (88 g/liter), pH to 7.0with 1M HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg/ml denaturedcalf thymus DNA at 65[deg.] C., (2) 1×SSC, 50% formamide, 1% SDS, 0.1-2mg/ml denatured calf thymus DNA at 42[deg.] C., (3) 1% bovine serumalbumin (fraction V), 1 mM Na2 . . . EDTA, 0.5M NaHP04 (pH 7.2) (1MNaHP04=134 g Na2HP04 . . . 7H20, 4 ml 85% H3P04 per liter), 7% SDS,0.1-2 mg/ml denatured calf thymus DNA at 65[deg.] C., (4) 50% formamide,5×SSC, 0.02M Tris-HCl (pH 7.6), I×Denhardfs solution (100×=10 g Ficoll400, 10 g polyvinylpyrrolidone, 10 g bovine serum albumin (fraction V),water to 500 ml), 10% dextran sulfate, 1% SDS, 0.1-2 mg/ml denaturedcalf thymus DNA at 42[deg.] C., (5) 5×SSC, 5×Denhardfs solution, 1% SDS,100 [mu]g/ml denatured calf thymus DNA at 65[deg.] C., or (6) 5×SSC,5×Denhardfs solution, 50% formamide, 1% SDS, 100 [mu]g/ml denatured calfthymus DNA at 42[deg.] C., with high stringency washes of either (1)0.3-0.1×SSC, 0.1% SDS at 65[deg.] C., or (2) 1 mM Na2EDTA, 40 mM NaHP04(pH 7.2), 1% SDS at 65[deg.] C. The above conditions are intended to beused for DNA-DNA hybrids of 50 base pairs or longer. Where the hybrid isbelieved to be less than 18 base pairs in length, the hybridization andwash temperatures should be 5-10[deg.] C. below that of the calculatedTm of the hybrid, where Tm in <0> C.=(2× the number of A and Tbases)+(4× the number of G and C bases). For hybrids believed to beabout 18 to about 49 base pairs in length, the Tm in <0> C.=(81.5[deg.]C.+16.6(log ioM)+0.41(% G+Q−0.61 (% formamide)−500/L), where “M” is themolarity of monovalent cations (e.g., Na<+>), and “L” is the length ofthe hybrid in base pairs.

Moderate stringency conditions can employ hybridization at either (1)4×SSC, (10×SSC=3M NaCl, 0.3M Na3-citrate . . . 2H20 (88 g/liter), pH to7.0 with 1M HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg/ml denaturedcalf thymus DNA at 65[deg.] C., (2) 4×SSC, 50% formamide, 1% SDS, 0.1-2mg/ml denatured calf thymus DNA at 42[deg.] C., (3) 1% bovine serumalbumin (fraction V), 1 mM Na2 . . . EDTA, 0.5M NaHP04 (pH 7.2) (1MNaHP04=134 g Na2HP04 . . . 7H20, 4 ml 85% H3P04 per liter), 7% SDS,0.1-2 mg/ml denatured calf thymus DNA at 65[deg.] C., (4) 50% formamide,5×SSC, 0.02M Tris-HCl (pH 7.6), I×Denhardfs solution (100×=10 g Ficoll400, 10 g polyvinylpyrrolidone, 10 g bovine serum albumin (fraction V),water to 500 ml), 10% dextran sulfate, 1% SDS, 0.1-2 mg/ml denaturedcalf thymus DNA at 42[deg.] C., (5) 5×SSC, 5×Denhardfs solution, 1% SDS,100 [mu]g/ml denatured calf thymus DNA at 65[deg.] C., or (6) 5×SSC,5×Denhardfs solution, 50%) formamide, 1% SDS, 100 [mu]g/ml denaturedcalf thymus DNA at 42[deg.] C., with moderate stringency washes ofI×SSC, 0.1% SDS at 65[deg.] C. The above conditions are intended to beused for DNA-DNA hybrids of 50 base pairs or longer. Where the hybrid isbelieved to be less than 18 base pairs in length, the hybridization andwash temperatures should be 5-10[deg.] C. below that of the calculatedTm of the hybrid, where Tm in <0> C.=(2× the number of A and Tbases)+(4× the number of G and C bases). For hybrids believed to beabout 18 to about 49 base pairs in length, the Tm in <0> C.=(81.5[deg.]C.+16.6(log ioM)+0.41(% G+Q−0.61 (% formamide)−500/L), where “M” is themolarity of monovalent cations (e.g., Na+), and “L” is the length of thehybrid in base pairs.

Low stringency conditions can employ hybridization at either (1) 4×SSC,(10×SSC=3M NaCl, 0.3M Na3-citrate . . . 2H20 (88 g/liter), pH to 7.0with 1M HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg/ml denaturedcalf thymus DNA at 50[deg.] C., (2) 6×SSC, 50% formamide, 1% SDS, 0.1-2mg/ml denatured calf thymus DNA at 40[deg.] C., (3) 1% bovine serumalbumin (fraction V), 1 mM Na2 . . . EDTA, 0.5M NaHP04 (pH 7.2) (1MNaHP04=134 g Na2HP04 . . . 7H20, 4 ml 85% H3P04 per liter), 7% SDS,0.1-2 mg/ml denatured calf thymus DNA at 50[deg.] C., (4) 50% formamide,5×SSC, 0.02M Tris-HCl (pH 7.6), IX Denhardfs solution (100×=10 g Ficoll400, 10 g polyvinylpyrrolidone, 10 g bovine serum albumin (fraction V),water to 500 ml), 10% dextran sulfate, 1% SDS, 0.1-2 mg/ml denaturedcalf thymus DNA at 40[deg.] C., (5) 5×SSC, 5×Denhardfs solution, 1% SDS,100 [mu]g/ml denatured calf thymus DNA at 50[deg.] C., or (6) 5×SSC,5×Denhardfs solution, 50%) formamide, 1%> SDS, 100 [mu]g/ml denaturedcalf thymus DNA at 40[deg.] C., with low stringency washes of either2×SSC, 0.1% SDS at 50[deg.] C., or (2) 0.5% bovine serum albumin(fraction V), 1 mM Na2EDTA, 40 mM NaHP04 (pH 7.2), 5% SDS. The aboveconditions are intended to be used for DNA-DNA hybrids of 50 base pairsor longer. Where the hybrid is believed to be less than 18 base pairs inlength, the hybridization and wash temperatures should be 5-10[deg.] C.below that of the calculated Tm of the hybrid, where Tm in <0> C.=(2×the number of A and T bases)+(4× the number of G and C bases). Forhybrids believed to be about 18 to about 49 base pairs in length, the Tmin <0> C.=(81.5[deg.] C.+16.6(log i0M)+0.41(% G+Q−0.61 (%formamide)−500/L), where “M” is the molarity of monovalent cations(e.g., Na.+), and “L” is the length of the hybrid in base pairs.

Immunogenic antigens can be produced recombinantly using a DNA sequencethat encodes the antigen, which has been inserted into an expressionvector and expressed in an appropriate host cell. DNA sequences encodingantigens can, for example, be identified by screening an appropriategenomic or cDNA expression library with sera obtained from patientsinfected with M. tuberculosis, HIV, Plasmodium, Leishmania, herpesvirus, or hepatitis virus. Such screens can generally be performed usingtechniques well known to those of ordinary skill in the art. Forexample, using oligonucleotide primers designed from above DNAsequences, PCR can be employed to isolate a gene from a cDNA or genomiclibrary.

Alternatively, genomic or cDNA libraries derived from M. tuberculosis,HIV, Plasmodium, Leishmania, herpes virus, or hepatitis virus can bescreened directly using peripheral blood mononuclear cells (PBMCs) or Tcell lines or clones derived from one or more immune individuals. Ingeneral, PBMCs and/or T cells for use in such screens can be prepared asdescribed below. Direct library screens can generally be performed byassaying pools of expressed recombinant proteins for the ability toinduce proliferation and/or interferon production in T cells derivedfrom an immune individual.

The invention also provides vectors, plasmids or viruses containing oneor more of the nucleic acid molecules (e.g., having the sequence of SEQID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35, 37, 39, 41, 43, 45, 47, or combinations thereof). Suitable vectorsfor use in eukaryotic and prokaryotic cells are known in the art and arecommercially available or readily prepared by a skilled artisan.

EXEMPLIFICATION Example 1

To generate recombinant S. mitis (r S. mitis), it was first establishedthat strain NCTC 12261 could be transformed and could express foreigngenes. The strategy was to express foreign vaccine antigens in thesecreted form, which is necessary for bacterial-expressed antigens to beimmunogenic. Thus far the genes encoding M. tuberculosis Ag85b and HIV-1HXBc2 env-His-tag in-frame have been successfully integrated in thegenome of S. mitis. These genes were synthetically produced with codonsoptimized for S. mitis. The genes integrated at the 5′ end of thepullulanase gene (pulA/Smt0163) encoding a signal peptide that allowsprocessing and secretion of the M. tuberculosis and HIV antigens. FIG. 2illustrates this integration strategy. The signal peptide has anamino-terminal region (N), a hydrophobic core (H), a signal peptidasecleavage site (C), and an accessory Sec transport motif (AST). Forstable expression, the M. tuberculosis Antigen 85b and HIV HXBc2 genesor the ermR cassette were integrated via homologous recombination intothe pulA gene of S. mitis. PulA was selected as the integration sitebecause the gene has a strong promoter and encodes localization motifsfor the gene product; furthermore pulA was not essential for bacterialgrowth. A DNA fragment containing ermR alone (control) or HIV and ermRor 85b and ermR flanked by 250 bp pulA 5′ and 3′ fragments wasconstructed and ligated into a suicide vector (pCR2.1) fortransformation and integration at the pulA locus (FIG. 3A). S. mitistransformants were selected on erythromycin-containing plates. To testwhether a foreign gene was integrated into S. mitis a genomic PCRanalysis was performed. PCR analysis showed that the S. mzYw-specificprimers A/B generated the expected 250 bp smt0163 PCR product, while theHIV-specific primer C and ermR-specific primer D generated the expected1 kb product (FIG. 4). In addition, S. mitis pulA specific primer X and85b-specific primer y generated the expected 250 bp product (FIG. 5,lanes 2-3).

Next, the expression of Env antigen in S. mitis using the C-terminalHis-tag fused to HIV antigen for detection was determined. An expressioncassette containing the HIV gene was then transformed into S. mitis forhomologous recombination into the pulA gene. S. mitis vaccine vectorcandidates were grown to mid-log phase in Todd-Hewitt Broth medium witherythromycin. Proteins from cell lysates (^g) and from concentratedsupematants (10 ml) were size fractionated by SDS-PAGE. HIV HXBc2 gpl20(Env) in the bacterial pellets and as a secreted product in thesupematants was detected by Western blotting using horseradishperoxidase conjugated anti-His tag antibody (Penta-His, Qiagen) and achemiluminescence substrate (Invitrogen). HIV Env linked to the PulAsignal peptide was expressed in S. mitis cell lysates of arepresentative recombinant clone (FIGS. 6A and 6B). Lysates alsocontained proteins that crossreact with the anti-His-tag antibody (FIG.6A). In addition, secreted Env was detected in bacterial culturesupematants (FIG. 5C). 100 ng of His-tagged M. tuberculosis protein(MT0401) was used as a positive control (FIGS. 6B and 6C, lane 1). Thearrow denotes the Env protein band present in the TCA-pecipitated (FIG.5C, lane 2) and Amicon-concentrated supernatant (FIG. 6C, lane 4),although not in acetone precipitates (FIG. 6C, lane 3). As expected, theHIV Env was not expressed in culture supematants of parent S. mitiscells (FIG. 6C, lanes 5-7). The apparent molecular weight was similar tothe expected molecular weight (of approximately 70 kDa), suggesting thatEnv was not glycosylated in S. mitis.

Recombinant S. mitis vector is stable after serial passages in vitro. Todetermine the stability of the integrated Env gene in r S. mitis, fourclones were picked at random and grown anaerobically for 24 hours whichrepresents approximately three generations (7.1 hours per generation) inTodd-Hewitt Broth (THB) without erythromycin. Cultures were diluted andgrown for another 24 hours for a total of approximately 6 generations.Cultures were diluted again and grown on THB plates withouterythromycin. From these plates, between 100 and 150 colonies werepicked at random and streaked to THB plates with and withouterythromycin. This procedure was repeated to measure the stability ofthe r S. mitis clones over approximately 30 generations on plates withand without antibiotic. Loss of erythromycin-resistance was taken as anindication of the loss of the Env transgene. The progeny from all fourclones were stable, with no loss of erythromycin resistance after tensubcultures or 30 generations of replication without antibiotic (FIG.7A). In addition, the expression of Env in the same daughter clones wasanalyzed. Western blot analysis revealed that they secreted the sameamount of Env antigen as the original r S. mitis clones; Env productionin a representative daughter clone after 30 generations and the originalHIVgpl20A clone is shown (FIG. 7B). These results indicate that theresulting r S. mitis is stable, a property that makes it a strongcandidate for further preclinical testing in animal models.

In summary, it was shown that S. mitis can undergo integrativetransformation and that the progenies express HIV-1 Env antigen and arehighly stable. These results demonstrate that we have successfullydeveloped a stable gene expression system in S. mitis for constructing ar S. mitis vaccine vector.

r S. mitis elicits HIV-specific immune responses in mice. 6-week oldBalb/c mice (4 mice per group) were inoculated i.p. twice (four weeksapart) with 108 cfu live r S. mitis expressing the HIV-1 HXBc2 Envprotein (r S. mitis HIVEnv), S. mitis with an intergrated ermr gene only(r S. mitis sham) or saline (Saline). One week following the secondinoculation, mice were bled retrorbitally to isolate peripheral bloodmononuclear cells (PBMC) which were then stimulated with either 5[mu]g/ml S. mitis soluble lysate or 5 [mu]g/ml recombinant HXBc2 proteinin vitro. Intracellular cytokine staining (ICS) was performed aspreviously described to assess T cell responses (7). Using the Flowjosoftware 7.6.3 (Tree Star), IFNy+, TNFa+, IL-2+, CD4+ or CD8+ T cellswere determined, and results from a representative mouse from each groupis shown (FIGS. 8 and 9). The Student's t test was used to determinecytokine response difference between the groups and a P value <0.5 wasconsidered significant. ICS analysis revealed that that PBMC from micevaccinated with the r S. mitis HIVEnv had statistically significanthigher CD4 T cell responses specific to S. mitis lysate antigenscompared to saline injected animals (Saline), producing more IFNy(P<0.003), TNFa (P<0.02) and IL-2 (P<0.002) (FIG. 6). The r S. mitisHIVEnv-vaccine group also generated higher frequency S. mitisspecific-CD8 T cells producing IFNy (P<0.02), and TNFa (P<0.004) andIL-2 (P<0.005) compared to the saline group (FIG. 8). With respect toHIV-specific T cell responses, the r S. mitis HIVEnv vaccinees producedmarked higher CD4 and CD8 T cell responses specific to the HIV envelopeprotein compared r S. mitis sham vaccine group. r S. mitis HIVEnvvaccinated mice generated higher frequency HIV Env-specific-CD4 T cellproducing IFNy (P<0.005), TNFa (P<0.02 and IL-2 (P<0.01) as well asHIV-specific-CD8 T cells producing IFNy (P<0.009), TNFa (P<0.03) andIL-2 (P<0.002) compared to r S. mitis sham-immunized mice (FIG. 9).Interestingly, r S. mitis was capable of inducing CD 8 T cell responsesin mice, which were detected after in vitro stimulation with S. mitislysate and full-length recombinant protein, which suggests thatexogenous S. mitis and HIV Env antigens were processed for crossprimingof CD8 T cells (12,16). This proof of concept immunogenicity study showsthat r S. mitis can induce S. mitis- and HIV-specific cellular immunity.

Example 2

Two candidate secretion/cell-wall anchored systems will be tested forthe expression of the HIV inhibitor, T20. Both the serine-richGspB-homologue or the pullulanase protein expressed in S. mitis will betested. Both proteins contain an N-terminal signal peptide (YSIRK type)for secretion and the LPXTG motif for cell-wall surface expression. Thesignal peptide determinants for secretion of GspB into culturesupernatants are the amino-terminal region (N), a hydrophobic core (H),a signal peptidase cleavage site (C), and the accessory Sec transport(AST). Interestingly, attachment of the signal peptide region butexclusion of the LPXTG sequence results in secretion of GspB in culturesupernatants. Hence, S. mitis expressing HIV proteins on the cell wallsurface will be constructed by flanking the viral protein with thesignal peptide and the LPXTG anchor, and S. mzYzs-secreting HIV proteinsor T20 inhibitor by attaching the signal peptide to the N-terminus.

A recombinant S. mitis secreting an HIV-1 inhibitor will be constructedas a microbicide since it was hypothesized that inhibiting HIV entryinto oral mucosal target cells will be a viable approach for preventingmother-to-child transmission. Recombinant S. mitis secreting the potentHIV peptide inhibitor, T20, will be constructed. T20 is a 36-amino acidpeptide (YTSLIHSLIEESQNQQEK EQELLELDKWASLWNWF (SEQ ID NO: 53)) that actsas a decoy a-helix and prevents HIV-1 infection by disrupting theassembly of the six-helix bundle viral fusion apparatus by mimicking theheptad repeat 2 (HR2) oligomerization domain of the gp41 envelopeglycoprotein. T20 is clinically proven to inhibit a diversity of primaryisolates of HIV-1 in humans. However, its delivery as a syntheticpeptide is associated with high cost and inconvenient dosing regimen,making this drug a reserve in patients with drug resistant HIV. Deliveryof this HIV inhibitor in the most cost-effective way will be highlybeneficial, and the approach to accomplish this is to deliver the T20inhibitor as an oral microbicide. Recombinant S. mitis secreting T20which can be administered orally will be generated. The T20 peptide willbe attached to N-terminal signal peptide of S. mitis pullulanase or GspBprotein homologue, and an IgA1 protease cleavage site will be added togenerate free T20 peptides. Engineered constructs will then be testedfor the ability to secrete T20 that will inhibit HIV using the standardneutralization assay (Cayabyab M, Karlsson G B, Etemad-Moghadam B A,Hofmann W, Steenbeke T, Halloran M, Fanton J W, Axthelm M K, Letvin N L,Sodroski J G: Changes in human immunodeficiency virus type 1 envelopeglycoproteins responsible for the pathogenicity of a multiply passagedsimian-human immunodeficiency virus (SHIV-HXBc2). J Virol 1999,73:976-984).

Example 3

Other commensal bacteria that encode a vaccine antigen can be generatedby using the genbank sequences below for the construction of a DNAfragment containing erythromycin resistance cassette that is flanked by250 bp pulA 5′ and 3′ fragments and ligating it into a suicide vector(pCR2.1) for transformation and integration at the pulA locus.

Example 4

A recombinant S. mitis was created to express a fusion construct (SEQ IDNO: 80) of the two homologous glucosylating exotoxins, TcdA and TcdB,produced by the pathogenic bacterium Clostridium difficile. The C.difficile toxin genes or the ermR cassette were integrated viahomologous recombination into the pulA gene of S. mitis. A DNA fragmentcontaining ermR alone (control) or C. difficile toxin and ermR flankedby 250 bp pulA 5′ and 3′ fragments was constructed and ligated into asuicide vector (pCR2.1) for transformation and integration at the pulAlocus (FIG. 3A). S. mitis transformants were selected onerythromycin-containing plates. To test whether a foreign gene wasintegrated into S. mitis a Western blot was performed. Anti-TCdA/TCdBmAb recognized the fusion construct in 3 different clones of therecombinant S. mitis construct but not in the wild-type S. mitis (FIG.18).

The genome of type strain S. mitis NCTC 12261 was sequenced and genbanknumbers can be accessed via pubmed. The Genbank # of S. mitis sequenceis as follows: Streptococcus mitis NCTC 12261 contig1, whole genomeshotgun sequence 113,379 bp linear DNAAccession:AEDX01000011.1GI:307615963 (SEQ ID NO: 55) Streptococcus mitisNCTC 12261 contig2, whole genome shotgun sequence 24,459 bp linear DNAAccession:AEDX01000017.1GI:307615908 (SEQ ID NO: 56) Streptococcus mitisNCTC 12261 contig3, whole genome shotgun sequence 81,409 bp linear DNAAccession:AEDX01000018.1GI:307615827 (SEQ ID NO: 57) Streptococcus mitisNCTC 12261 contig4, whole genome shotgun sequence 314,111 bp linear DNAAccession:AEDX01000019.1GI:307615569 (SEQ ID NO: 58) Streptococcus mitisNCTC 12261 contig5, whole genome shotgun sequence 100,480 bp linear DNAAccession:AEDX01000020.1GI:307615480 (SEQ ID NO: 59) Streptococcus mitisNCTC 12261 contig6, whole genome shotgun sequence 91,167 bp linear DNAAccession:AEDX01000021.1GI:307615386 (SEQ ID NO: 60) Streptococcus mitisNCTC 12261 contig7, whole genome shotgun sequence 84,457 bp linear DNAAccession:AEDX01000022.1GI:307615313 (SEQ ID NO: 61) Streptococcus mitisNCTC 12261 contig8, whole genome shotgun sequence 111,153 bp linear DNAAccession:AEDX01000023.1GI:307615211 (SEQ ID NO: 62) Streptococcus mitisNCTC 12261 contig9, whole genome shotgun sequence 112,014 bp linear DNAAccession:AEDX01000024.1GI:307615105 (SEQ ID NO: 63) Streptococcus mitisNCTC 12261 contig10, whole genome shotgun sequence 88,437 bp linear DNAAccession:AEDX01000001.1GI:307616687 (SEQ ID NO: 64) Streptococcus mitisNCTC 12261 contig11, whole genome shotgun sequence 93,022 bp linear DNAAccession:AEDX01000002.1GI:307616595 (SEQ ID NO: 65) Streptococcus mitisNCTC 12261 contig12, whole genome shotgun sequence 71,904 bp linear DNAAccession:AEDX01000003.1GI:307616529 (SEQ ID NO: 66) Streptococcus mitisNCTC 12261 contig13, whole genome shotgun sequence 164,416 bp linear DNAAccession:AEDX01000004.1GI:307616373 (SEQ ID NO: 67) Streptococcus mitisNCTC 12261 contig14, whole genome shotgun sequence 75,285 bp linear DNAAccession:AEDX01000005.1GI:307616290 (SEQ ID NO: 68) Streptococcus mitisNCTC 12261 contig15, whole genome shotgun sequence 16,944 bp linear DNAAccession:AEDX01000006.1GI:307616272 (SEQ ID NO: 69) Streptococcus mitisNCTC 12261 contig16, whole genome shotgun sequence 112,411 bp linear DNAAccession:AEDX01000007.1GI:307616176 (SEQ ID NO: 70) Streptococcus mitisNCTC 12261 contig17, whole genome shotgun sequence 22,040 bp linear DNAAccession:AEDX01000008.1GI:307616156 (SEQ ID NO: 71) Streptococcus mitisNCTC 12261 contig18, whole genome shotgun sequence 124,845 bp linear DNAAccession:AEDX01000009.1GI:307616054 (SEQ ID NO: 72) Streptococcus mitisNCTC 12261 contig19, whole genome shotgun sequence 2,436 bp linear DNAAccession:AEDX01000010.1GI:307616052 (SEQ ID NO: 73) Streptococcus mitisNCTC 12261 contig20, whole genome shotgun sequence 8,218 bp linear DNAAccession:AEDX01000012.1GI:307615955 (SEQ ID NO: 74) Streptococcus mitisNCTC 12261 contig21, whole genome shotgun sequence 5,961 bp linear DNAAccession:AEDX01000013.1GI:307615946 (SEQ ID NO: 75) Streptococcus mitisNCTC 12261 contig22, whole genome shotgun sequence 4,520 bp linear DNAAccession:AEDX01000014.1GI:307615941 (SEQ ID NO: 76) Streptococcus mitisNCTC 12261 contig23, whole genome shotgun sequence 3,240 bp linear DNAAccession:AEDX01000015.1GI:307615936 (SEQ ID NO: 77) Streptococcus mitisNCTC 12261 contig24, whole genome shotgun sequence 4,773 bp linear DNAAccession:AEDX01000016.1GI:307615932 (SEQ ID NO: 78) Streptococcus mitisNCTC 12261, whole genome shotgun sequencing project 1,831,081 bp otherDNA.

This entry is the master record for a whole genome shotgun sequencingproject and contains no sequence data.Accession:NZ_AEDX00000000.1GI:307708845 Streptococcus mitis NCTC 12261,whole genome shotgun sequencing project 24 rc linear DNA. This entry isthe master record for a whole genome shotgun sequencing project andcontains no sequence data. Accession:AEDX00000000.1GI:307616765

The relevant teachings of all the references, patents and/or patentapplications cited herein are incorporated herein by reference in theirentirety. While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed:
 1. An integrally transformed non-pathogenic, commensalbacterium that expresses a synthetic nucleic acid molecule encoding aforeign fusion polypeptide comprising a C. difficile antigen and asignal polypeptide therein, wherein the synthetic nucleic acid moleculeis stably integrated into genomic DNA of the bacterium, wherein thebacterium is Streptococcus mitis, and wherein the synthetic nucleic acidmolecule comprises a TcdA/TcdB fusion construct having SEQ ID NO:81. 2.The integrally transformed non-pathogenic, commensal bacterium of claim1, wherein the foreign fusion polypeptide elicits an immunogenicresponse in an individual.
 3. The integrally transformed non-pathogenic,commensal bacterium of claim 1, wherein the foreign fusion polypeptideis expressed in the cytoplasm, in the cell membrane or cell wall, orexports to the cell surface of the bacterium.
 4. The integrallytransformed non-pathogenic, commensal bacterium of claim 3, wherein thesignal polypeptide is selected from the group consisting of: serine-richGspB homologue and the pullulanase polypeptide.
 5. The integrallytransformed non-pathogenic, commensal bacterium of claim 1, wherein theTcdA/TcdB fusion construct encodes an amino acid sequence comprising SEQID NO:
 82. 6. The integrally transformed non-pathogenic, commensalbacterium of claim 5, wherein the TcdA/TcdB fusion construct is insertedinto a gene that encodes a pullulanase polypeptide.
 7. The foreignfusion polypeptide expressed by the integrally transformednon-pathogenic, commensal bacterium of claim
 1. 8. A method ofdelivering a foreign fusion polypeptide to an individual comprising:contacting the integrally transformed non-pathogenic, commensalbacterium of claim 1 with tissue of the oral cavity or upper respiratorytract of the individual in an amount sufficient for colonization of saidbacterium.